An automated high-content synaptic phenotyping platform in human neurons and astrocytes reveals a role for BET proteins in synapse assembly

Abstract

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth and cell viability, validating six small molecules which specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression programs. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

“…Finally, astrocytes play critical roles in the establishment of synaptic networks, a key non-cell-autonomous function of astrocytes in the human brain. Previous studies have shown that both rodent and human astrocytes can improve the maturation of hNs (Berryer, 2022;Canals et al, 2018;Hedegaard et al, 2020;Nehme et al, 2018). Using an established glutamatergic hN differentiation protocol (Nehme et al, 2018) combined with an automated synaptic quantification platform (Berryer, 2022), we analyzed synaptic development in hN + hiA cocultures derived from the same parental cell line, as well as hN + hpA co-cultures.…”

“…Human induced neuron (hN) generation, astrocyte co-culture and synapse quantification was performed as previously described (Berryer, 2022;Nehme et al, 2018;Zhang et al, 2013). In brief, hNs with stable integration of iNGN2 in the AAVS1 safe-harbor locus were differentiated on day 0 in N2 medium supplemented with SB431542, XAV939 and LDN-193189 along with doxycycline and Y27632.…”

“…To generate hiAs, a doxycycline inducible NGN2 expression construct was introduced into hPSCs through TALEN-mediated stable integration into the AAVS1 safe-harbor locus (Berryer, 2022). Following neomycin selection for iNGN2 integration, iNGN2-hPSCs were briefly switched to N2 medium with doxycycline and small molecule patterning for 48hrs to induce a forebrain NPC-like fate (Nehme et al, 2018) (Fig.…”

“…Previous studies have shown that both rodent and human astrocytes can improve the maturation of hNs (Berryer, 2022;Canals et al, 2018;Hedegaard et al, 2020;Nehme et al, 2018). Using an established glutamatergic hN differentiation protocol (Nehme et al, 2018) combined with an automated synaptic quantification platform (Berryer, 2022), we analyzed synaptic development in hN + hiA cocultures derived from the same parental cell line, as well as hN + hpA co-cultures. These analyses revealed significant increases in the density and the area of presynaptic puncta, identified by SYNAPSIN1 aggregates localized on and along the MAP2-expressing neurites in hiA + hN as compared to hpA + hN co-culture, with no difference in the area occupied by MAP2 positive neurites or the number of DAPI positive nuclei detected.…”